Malignant glioma, in particular, polymorphism glioblastoma (GBM) is the most common and desperate form of the primary brain tumor of adults (for example, see Non-Patent Document 1). Since GBM tends to infiltrate widely into normal brain tissues in the periphery of GBM, it is virtually impossible to completely remove GBM even with the assistance of recent neural image processing techniques and improved surgical techniques. In the situation, another type of therapy has been required. However, despite great progress has been made in diagnostic procedure, radiation therapy, chemotherapy and supportive therapy, these neoplasms show strong resistance to the conventional therapies (for example, see Non-Patent Documents 2, 3, 4 and 5). The prognosis of the patients remains poor for the past 20 years. Most of the patients succumb to the disease within one year from the diagnosis (for example, see Non-Patent Documents 6 and 7). Since there are no effective therapies to be applied to the disease and patient's prognosis is extremely poor, options for a new therapy such as gene therapy must be offered.
Up to present, tumor transduction (gene introduction into a tumor) by means of a replication-defective vector has not yet been achieved with high efficiency. In the circumstances, a replication-competent virus vector is expected to serve as a cancer therapeutic agent (for example, see Non-Patent Documents 8, 9 and 10). The recent development in gene therapy for cancer has been made primarily on the use of an oncolytic virus, which not only delivers a cytotoxic gene to tumor cells but also directly disrupts the tumor cells through lytic-infection as well as designed so as to selectively replicate in the dividing cells by modifying the gene thereof (for example, see Non-Patent Documents 11 and 12). With respect to at least ten types of virus species, clinical tests have started (see Non-Patent Document 13). Use of an oncolytic virus designed so as to selectively replicate in dividing cells has been greatly expected from oncolytic activity and safety, since the effect of an anticancer agent injected to tumor mass can be anatomically enlarged by intraneoplastic replication and the oncogenic effect can be augmented by delivering an anticarcinogenic gene (for example, see Non-Patent Documents 14 and 15).
HSV is a double stranded virus, which has the longest genome (153 kb) of the DNA viruses proliferating within the nucleus and encodes 84 types of open reading frames. The genome is composed of an L (Long) region and an S (Short) region and having unique sequences each flanked by inverted repeat sequences. The entire base sequence of the virus genome has been determined and almost all functions of the genes of the virus have been elucidated. As an oncolytic mutant of HSV designed so as to selectively replicate in the dividing cells, two types of HSV mutants having a single gene mutated have been investigated. One of the mutants is a virus mutant, which has a defect in the function of the viral gene required for nucleic acid metabolism, such as thymidine kinase, ribonucleotide reductase (RR) or uracil N-glycosylate. The other one is a virus mutant, which has a defect in the function of γ34.5 (ICP34.5) gene serving as a virulence factor by increasing significantly the number of viruses released from an infected cell by inhibiting interrupt of protein synthesis of a host (for example, see Non-Patent Documents 16 and 17).
The HSV mutant having a single gene mutation has a risk of going back to a wild type through back mutation due to homologous replication or the like. Other than this, there are the following problems. For example, in a thymidine kinase function defective mutant, resistance to ganciclovir may increase. In a γ34.5 function defective mutant, oncolytic activity may decrease. In the circumstances, in order to reduce the risk of back mutation to a wild type, an HSV mutant having a plurality of mutations introduced therein has been developed. Examples of such an HSV mutant may include G207 and MGH1 defective in two functions: a γ34.5 defective mutation and a ribonucleotide reductase insertional mutation. When such a double mutant is directly injected to the skull, it replicates relatively selectively in tumor cells compared to normal tissue cells while significantly decreasing neurovirulence and maintaining sensitivity to ganciclovir. Since such a double mutant HSV lineage maintains a defective γ34.5 gene, it is virulent to normal tissue but weak. Conversely, these mutants apparently show an oncolytic effect upon tumor cells; however, the oncolytic effect thereof is lower than that produced by a mutant having non-defective γ34.5 gene.
The mutant G207 was developed by Martuza et al. as a double mutant of herpes simplex virus type I (HSV-I) by introducing a deletion in the γ34.5 gene and inserting the LacZ gene into the ribonucleotide reductase (ICP6) gene (for example, see Non-Patent Document 18, Patent Document 1). The mutant G207 is superior to other viruses in view of therapy. The mutant G207 is reproduced in dividing cells, with the result that the infected cells causes cell lysis to die. However, the proliferation of G207 is significantly attenuated in non-dividing cells. When G207 is injected into a tumor established in an athymic mouse, growth of the tumor is suppressed by tumor specific replication. As a result, the life of the mouse is prolonged (for example, see Non-Patent Document 19). Furthermore, when G207 is injected into a tumor in an immune responsive mouse, a tumor specific immune response is induced, with the result that proliferation of a tumor in which G207 is not injected therein is also suppressed (for example, see Non-Patent Document 20). In this case, G207 serves as an in-situ cancer vaccine. Up to present, gene therapy using mutant herpes virus G207 has been applied primarily to brain tumors and clinical application of G207 has been started in the United State (for example, see Non-Patent Document 9). In the clinical application, the safety of G207 has been proven; however, it has been reported that G207 is not so effective according to the clinical results so far reported (for example, see Non-Patent Documents 9 and 10).
When a viral vector is constructed for clinical application, the viral vector must have both safety and oncolytic efficacy (for example, see Non-Patent Documents 21 and 22). The safety varies directly depends upon the replication selectiveness of a virus mutant, whereas the efficacy varies depending upon the ability of the virus to effectively transmit tumor cells and proliferate in the neoplastic mass infected. An HSV vector having a plurality of mutations, such as G207, has been constructed with effort in an attempt to improve safety and reduce the risk of back mutation to a wide type. However, their oncolytic efficacy seems to decrease, at the same time. One of defective genes in G207 is γ34.5 gene, which is also called a neurovirulence factor. The γ34.5 gene has a role in significantly increasing the number of viral cells released from an infected cell (host) by inhibiting interrupt of host protein synthesis (for example, see Non-Patent Documents 16 and 23). G207 is greatly improved in safety by deleting the γ34.5 gene; however, it seems that the therapeutic effect has slightly decreased.
Several strategies are known to construct an HSV selectively replicating in a tumor cell. For example, mention may be made of making a deletion or mutation of a virus gene required for replication of a division-completed cell (for example, see Non-Patent Documents 12, 14 and 24); making a deletion of a virus gene responsible for regulating production of viral progenies (for example, see Non-Patent Documents 25, 26 and 27); using a tumor specific promoter for regulating expression of an indispensable virus gene (for example, see Non-Patent Document 28); modifying receptor specificity of a HSV glycoprotein to a tumor rather than normal tissues (for example, see Non-Patent Document 29), and so forth.
Musashi1 is a neural RNA binding protein found by the present inventors and is an evolutionally well conserved marker for a neural stem cell/precursor cell (for example, see Non-Patent Documents 30, 31, 32 and 33). Musashi1 has a high possibility of playing a crucial role in post-transcriptional gene regulation, which is essential for proper development of neural cells and glia cells (for example, see Non-Patent Documents 30, 31, 32, 33 and 34). Recent studies have elucidated that Musashi1 is expressed in a plurality of tumors, especially, a malignant glioma in the central nerve system (for example, see Non-Patent Documents 35, 36 and 37) and can be used as a marker for a malignant glioma (for example, see Non-Patent Documents 36 and 37). Other studies have reported that the mouse Musashi1 promoter (P/Musashi1) works in the human fetus brain, and that neural stem cells can be screened by a fluorescence activated cell sorter (FACS) based on GFP expression driven by P/Musashi1 (for example, see Non-Patent Document 38).
As is described above, clinical tests of an oncolytic virus therapy using HSV-G207 for glioma are carrying out in the United States. The safety of HSV-G207 has been demonstrated; however, the therapeutic effect has not yet been proved. This is conceivably because G207 is attenuated, leading to a replication attenuation type. Then, the present inventors came up with an idea that if the γ34.5 gene inactivated in G207 is reproduced in a glioma alone by use of the Musashi1 promoter selectively working in the glioma, stronger therapeutic effect can be exerted. Then, they developed an amplicon vector expressing γ34.5 by the Musashi1 promoter (Patent Document 2). However, since a process for producing the amplicon vector is complicated, it was difficult to obtain a stable amplicon vector. Thus, it was not easy to put it into clinical use.
Patent Document 1: U.S. Pat. No. 5,585,096
Patent Document 2: Japanese Patent Laid-Open No. 2005-73653
Non-Patent Document 1: Benign Cerebral Gliomas, Vol. 1. pp. 181-189, 1995
Non-Patent Document 2: Hum. Gene Ther. 8, 965-977, 1997
Non-Patent Document 3: Pathol. Res. Pract. 194, 149-155,
Non-Patent Document 4: Proc. Natl. Acad. Sci. U.S.A. 95, 14453-14458, 1998
Non-Patent Document 5: J. Neurooncol. 42, 95-102, 1999
Non-Patent Document 6: J. Neurosurg. 88, 1-10, 1998
Non-Patent Document 7: Cancer Res. 62, 756-763, 2002
Non-Patent Document 8: Nat. Med. 6,879-885, 2000
Non-Patent Document 9: Gene Ther. 7,867-874, 2000
Non-Patent Document 10: Gene Ther. 7,859-866, 2000
Non-Patent Document 11: Surg. Oncol. Clin. N. Am. 7, 589-602, 1998
Non-Patent Document 12: Science 252, 854-856, 1991
Non-Patent Document 13: Nat. Med. 7, 781-787, 2001
Non-Patent Document 14: Hum. Gene. Ther. 5, 183-191, 1994
Non-Patent Document 15: Cancer Res. 58, 5731-5737, 1998
Non-Patent Document 16: Proc. Natl. Acad. Sci. U.S.A. 89, 3266-3270, 1992
Non-Patent Document 17: Nat Cell Biol 3, 745-750, 2001
Non-Patent Document 18: Nat. Med. 1, 938-943, 1995
Non-Patent Document 19: Cancer. Res. 55, 4752, 1995
Non-Patent Document 20: Hum. Gene Ther. 9, 2177-2185, 1998
Non-Patent Document 21: J. Virol. 73, 3843-3853, 1999
Non-Patent Document 22: J. Virol. 74, 4765-4775, 2000
Non-Patent Document 23: Science 250, 1262-1266, 1990
Non-Patent Document 24: Hum. Gene Ther. 8, 533-544, 1997
Non-Patent Document 25: Neurosurgery 32, 597-603, 1993
Non-Patent Document 26: Proc. Natl. Acad. Sci. USA 92, 1411-1415, 1995
Non-Patent Document 27: Lab. Investig. 73, 636-648, 1995
Non-Patent Document 28: J. Virol. 73, 7556-7564, 1999
Non-Patent Document 29: J. Virol. 72, 9683-9697, 1998
Non-Patent Document 30: Neuron 13, 67-81, 1994
Non-Patent Document 31: Dev. Biol. 176, 230-242, 1996
Non-Patent Document 32: Genomics 52, 382-384, 1998
Non-Patent Document 33: Dev. Neurosci. 22, 139-153, 2000
Non-Patent Document 34: J. Neurosci. 17, 8300-8312, 1997
Non-Patent Document 35: Differentiation. 68, 141-152, 2001
Non-Patent Document 36: BBRC 293, 150-154, 2002
Non-Patent Document 37: GLIA 34, 1-7, 2001
Non-Patent Document 38: Nat. Biotechnol. 19, 843-850, 2001